JPH0112760B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for concentrating and purifying interferon. Interferon is a protein produced by living cells by stimulating them with a virus or special agent, and its chemical structure differs depending on the animal species, and even in the same animal species, multiple interferons are produced. Elon species are known. It also varies somewhat depending on the type of producing cell. Interferons recovered from cells or extracellular fluids produce protective effects against a wide range of viruses when treated with animal tissues or cells. Interferon has antiviral activity that is nonspecific to virus species and has effective antiviral activity against tissues and cells of the same species as the cells that produced interferon, but has a high antiviral activity against tissues and cells of other species. It is known to be species-specific in that it is ineffective or only provides weak antiviral activity in some cases. In addition to its antiviral activity, interferon is known to have antitumor properties, cell proliferation inhibitory effects, promotion of macrophage function, and other multifaceted effects. (“Advances in Interferon Research” (Protein Nucleic Acid Enzyme Special Issue No. 25,
Kyoritsu Shuppan, 1981), Shigeyasu Kobayashi, "Expanded edition of Interferon - Prospects from basic research to clinical application"
(1978, Kodansha Scientific), etc. Therefore, interferon is expected to be used as a therapeutic or preventive drug for viral diseases such as hepatitis B, herpes, and influenza, and as an anticancer agent for brain tumors, osteosarcoma, leukemia, and other diseases. Here, the interferon to be administered to humans must be human interferon because of the species specificity of interferon mentioned above, which is naturally produced by human cells. Currently, human interferon is classified into three types: human interferon α, β, and γ. Human interferon α is represented by interferon produced by human white blood cells, and human interferon β is represented by interferon (human fibroblast interferon) produced by human diploid fibroblasts. be done. Human interferon gamma is also an interferon produced by T lymphocytes. The present invention relates to a method for concentrating and purifying human interferon, and particularly provides an optimal method for concentrating and purifying human interferon β. Human interferon β is usually produced by human interferon β producing cells (hereinafter sometimes simply referred to as cells) cultured on a surface such as glass or plastic or on a microcarrier surface of DEAE-modified dextran. After induction treatment with synthetic double-stranded RNA such as
By culturing for ~48 hours, human interferon β is produced in this culture solution and obtained as a production solution containing human interferon β. In this case, as the culture solution, for example, Eagle
MEM culture medium is used, and serum and various additives are added as necessary. Of course, other culture solutions may be used. The human interferon in the production solution containing human interferon obtained in this way is generally at a low concentration [several thousand to tens of thousands of units (by biological assay, it is determined by the NIH-provided human interferon rebalance standard). This product is used for medical purposes because it contains many impurities derived from cells, culture, or additives in addition to human interferon. It is required to concentrate and purify human interferon. The biological activity of pure human interferon is said to be 0.4 to 1×10 ⁹ units per mg of protein. Many methods have been reported for enriching and purifying human interferon. For example, a precipitation method using ammonium sulfate, a membrane concentration method using a hollow fiber ultrafiltration membrane, an ion exchange method using CM-Sephadex, a molecular sieving method using Sephadex, polyacrylamide gel, etc. are known. Utilizing the hydrophobicity of human interferon β, there is also a method for concentrating and purifying human interferon by hydrophobic chromatography using a hydrophobic carrier such as concanavalin A, an octyl group, a tryptopyl group, a phenyl group, etc., introduced into agarose gel. Are known. [WA Carter et al., Biochemistry
15, 704 (1976); J.Biol.Chem. 251 , 5381 (1976);
ibid. 251 , 7260 (1976)]. In recent years, it has been known to purify interferon using a carrier bound with a dye having the chemical structure shown below. This pigment is a blue pigment sold by CIBA-GEIGY under the trade name "Cibacron Blue F3GA" or "Cibacron Blue 3GA", and its general name is CI Reactive Blue 2. Purification and characterization of human and animal interferon using carriers bound with the above dyes
Jankowski et al., Biochemistry, Vol. 15, pp. 5182-5187 (1976), and Bollin et al., Preparative Biochemistry, Vol.
292 (1978) and by Erickson et al. in Archives of Virology, Vol. 63, pp. 253-261 (1980). In JP-A-50-61997, E. Knight et al. used a crude human interferon β solution to bind the above dye to a cross-linked agarose gel to produce "Bluecephalose".
(manufactured by Pharmacia) with specific activity of 4 x 10 ⁸ units/
The company claims to have obtained purified human interferon-β with a purity of 95% or higher in mg/protein at a yield of 50%. However, in order to use the purified human interferon β as an injectable product, a so-called desalting process is required to remove the ethylene glycol and high concentration of sodium chloride contained therein. For desalting, there are methods such as gel filtration using "Sephadex G-25" or dialysis, but since ethylene glycol has high viscosity, it is difficult to remove it sufficiently. Furthermore, simply purifying with "Blue Cepharose" does not necessarily remove pyrogens sufficiently, which poses a problem for use in injections. Furthermore, "Bluecephalus" has an affinity for actinomycin D used in the production of interferon, and actinomycin D is likely to be mixed into the interferon recovery section. Although actinomycin D is used as an anticancer drug, it is also a strong mutagen, and its contamination is an extremely serious problem. Furthermore, V.
The method of G. Edy et al. is known [J. Biol. Chem.
252, 5934 (1978)]. Edy et al. reported that the crude F-IF solution (20,000 IU/
ml, protein content 0.4 mg/ml) was left on a column under neutral conditions overnight to adsorb F-IF onto the column, and then an acidic eluate was used to obtain a 32-fold purified solution of F-IF. . In addition, 10 ⁸ per mg of protein was purified 3100 times using the eluate PH gradient method.

Claims (1)

[Claims] 1. Contact the crude human interferon β solution with an insoluble blue carrier in which CI Reactive Blue 2 and crosslinked agarose gel are linked by ether bonds, and after adsorbing the interferon to the blue carrier, use the eluate. The interferon is recovered as a solution, and then the interferon recovery liquid is brought into contact with a chelate group-bonded carrier chelated with at least one metal selected from the group consisting of cobalt, nickel, and zinc, and the interferon is recovered as a solution. After adsorption to the chelate group-bonded carrier,
A method for concentrating and purifying human interferon β, which comprises recovering the interferon as a solution using an eluate.